Active-site titration of glycosyl hydrolases

ABSTRACT

The invention provides a method of screening for a property of a glycosyl hydrolase wherein the property is dependent on the concentration of the glycosyl hydrolase. The method comprises a step for determining the concentration of the glycosyl hydrolase by active site titration.

BACKGROUND

When libraries of enzymes are screened for properties which aredependent on the concentration of the enzyme, such as specific activity,it is essential to have available a fast and reliable method fordetermining the concentration of the enzyme. It is an advantage if themethod can be implemented in a standard microtiter plate based screeningsetup. The method must also be fast enough not to be a major bottleneckin high throughput screening. Further it must be capable of determiningthe concentration of the enzyme based on very small volumes of enzymesolution—e.g. less than the volume of a well in a microtiter plate. Thepresent invention has all the above-mentioned advantages.

SUMMARY

The present invention discloses a method for determining theconcentration of glycosyl hydrolases, which can form part of a screeningsetup. Accordingly, as a first aspect, the invention provides a methodfor determining the concentration of a glycosyl hydrolase by active-sitetitration using an inhibitor having a K_(d) which is at least 25 timeslower than the concentration of glycosyl hydrolase or, when the glycosylhydrolase is a retaining glycosyl hydrolase, using a substrate whereinthe rate constant for the glycosylation step is at least 10 times largerthan for the deglycosylation step.

In a second aspect, the invention provides a method of screening for aproperty of a glycosyl hydrolase wherein the property is dependent onthe concentration of the glycosyl hydrolase, comprising the steps of:

-   a) arranging a population of host cells expressing glycosyl    hydrolases in a spatial array wherein each position of the spatial    array is occupied by one or more cells expressing a specific    glycosyl hydrolase,-   b) cultivating the host cells in a suitable growth medium,-   c) determining the concentration of the glycosyl hydrolase of each    position of the spatial array by active-site titration using an    inhibitor having a K_(d) which is at least 25 times lower than the    concentration of glycosyl hydrolase or, when the glycosyl hydrolase    is a retaining glycosyl hydrolase, using a substrate wherein the    rate constant for the glycosylation step is at least 10 times larger    than for the deglycosylation step,-   d) assaying the glycosyl hydrolase of each position of the spatial    array for the property and relating the result to the concentration.

DETAILED DESCRIPTION

Active Site Titration Using Tight-Binding Inhibitor

Active concentration of an enzyme can be determined if a suitableinhibitor is available. The inhibitor should react with the enzyme witha known stochiometric ratio, preferably 1:1. The inhibitor-enzymecomplex should have reduced activity compared to uncomplexed enzyme witha given substrate; preferably the inhibitor-enzyme complex should beinactive.

The affinity of the inhibitor for the enzyme should be sufficientlystrong to assure that only insignificant amount of free inhibitor ispresent when inhibitor is mixed with a surplus of enzyme, i.e. foractive site titration to be applicable, the dissociation constant Kd forthe reaction:E+I←→EIwhere E is free enzyme, I is free inhibitor, EI is the enzyme-inhibitorcomplex, and K_(d)=[E]*[I]/[EI] at equilibrium, should be at least 25times, preferably at least 50 times, more preferably at least 100 times,most preferably at least 500 times, and in particular at least 1000times lower than the enzyme concentrations used. This requirement ofcourse includes inhibitors where reaction with enzyme is irreversible.

The inhibitor should be specific, i.e. binding of inhibitor to othercompounds in the enzyme solution should be insignificant (i.e. eitherthe concentration of these other compounds with reactivitytowards/affinity for the inhibitor should be much lower than the enzymeconcentration or their reactivity towards/affinity for the inhibitorshould be low).

Normally, active site titration of an enzyme solution will be done bymixing at least two (preferably more) aliquots of the enzyme solutionwith various suitable amounts of inhibitor. The mixtures of inhibitorand enzyme are incubated under conditions assuring that reaction betweeninhibitor and enzyme gets sufficiently close to equilibrium. At leasttwo (preferably more) inhibitor concentrations below the equivalencepoint with enzyme should be used. Subsequently, residual enzymeactivities in the inhibitor/enzyme mixtures are measured using asuitable substrate. Preferably, the substrate should be unable to affectthe equilibrium between inhibitor and enzyme significantly. This cane.g. be accomplished by using the substrate at concentrations much lowerthan the Michaelis-Menten constant K_(m) or by assuring that theincubation time with substrate is short compared to the dissociationrate for the enzyme-inhibitor complex.

Active Site Titration Using Burst Titration with Specific Substrate

Hydrolysis by retaining glycosyl hydrolases can be described by thereaction scheme:

where E is the enzyme, S is the substrate, ES′ is a glycosyl-enzymeintermediate formed by the glycosylation step, P₁ is the productreleased in the glycosylation step corresponding to the fragment withthe newly formed non-reducing end or the aglycon, P₂ is the productreleased in the deglycosylation step corresponding to the fragment withthe newly formed reducing end, and k₁, k₋₁, k₂ and k₃ are reaction rateconstants.

Assuming quasi steady-state for the concentration of the intermediate ESand that the used substrate concentration [S] is much larger than thetotal enzyme concentration [E]_(tot) and therefore approximatelyconstant during the experiment, integration of the differential equationfor the formation of product P₁ gives:$\left\lbrack P_{1} \right\rbrack = {{\frac{\frac{k_{2} \cdot k_{3}}{k_{2} + k_{3}} \cdot \lbrack S\rbrack \cdot \lbrack E\rbrack_{tot}}{K_{M} + \lbrack S\rbrack} \cdot t} + {\left( \frac{\frac{k_{2}}{k_{2} + k_{3}}}{1 + \frac{K_{M}}{\lbrack S\rbrack}} \right)^{2} \cdot \lbrack E\rbrack_{tot} \cdot \left( {1 - {\mathbb{e}}^{{- {(\frac{K_{M} \cdot {\lbrack S\rbrack}}{\frac{K_{M}}{k_{3}} + \frac{\lbrack S\rbrack}{k_{2} + k_{3}}})}} \cdot t}} \right)}}$where K_(M) is the observed Michaelis-Menten constant when quasi-steadystate is reached for the intermediate ES′ and given by:$K_{M} = \frac{\left( {k_{- 1} + k_{2}} \right) \cdot k_{3}}{k_{1} \cdot \left( {k_{2} + k_{3}} \right)}$

As seen from the equation for formation of P₁, the initial burst will befollowed by a phase where formation of P₁ is approximately linear andgiven by:$\left\lbrack P_{1} \right\rbrack = {{{\frac{\frac{k_{2} \cdot k_{3}}{k_{2} + k_{3}} \cdot \lbrack S\rbrack \cdot \lbrack E\rbrack_{tot}}{K_{M} + \lbrack S\rbrack} \cdot t} + {\left( \frac{\frac{k_{2}}{k_{2} + k_{3}}}{1 + \frac{K_{M}}{\lbrack S\rbrack}} \right)^{2} \cdot \lbrack E\rbrack_{tot}}} = {{\alpha \cdot t} + \beta}}$

The intercept β is seen to be approximately equal to the total enzymeconcentration [E]_(tot) if the rate constant k₂ is much larger than therate constant k₃ and the substrate concentration [S] is much larger thanthe Michaelis-Menten constant K_(M).

Thus, burst titration requires a substrate where the rate constant forthe glycosylation step k₂ is at least 10 times, preferably at least 50times, more preferably at least 100 times, most preferably at least 500times, and in particular at least 1000 times larger than the rateconstant for the deglycosylation step k₃ and the product P₁ isdetectable. The substrate concentration should be at least 10 times,preferably at least 100 times the concentration of the enzyme. Also, thesubstrate concentration should preferably be at least 10 times theMichaelis-Menten constant K_(M), otherwise the release of P₁ should bemeasured with at least two different substrate concentrations. Withthese requirements fulfilled, the total enzyme concentration [E]_(tot)can be found by fitting measured concentrations of P₁ to the equationsabove.

Inhibitors/Substrates

One class of inhibitors according to the invention, which are suitablefor determining the concentration of glucoamylases (and otheralpha-glucosidases) comprise acarbose and homologous thereof. All thesepseudo-oligosaccharide inhibitors have an acarviosine moiety at thenon-reducing end with various sugars attached to the reducing end. Inacarbose, maltose is attached to the acarviosine. The resemblance of theplanar structure of the hydroxymethylconduritol unit at the non-reducingend of acarbose to the transition state for hydrolysis of maltodextrinsresults in tight binding to the active site of glucoamylase, and the lowreactivity of the N-glucosidic linkage between thehydroxymethylconduritol residue and the4,6-dideoxy-4-amino-D-glucopyranose residue assures that the acarbose innot hydrolysed.

Other examples of inhibitors which may successfully be used in thepresent invention, include, but are not limited to:

tendamistat,

trestatin,

oligostatin,

nojirimycin and 1-deoxy-nojirimycin,

pyridinolol,

various isoflavinoids,

panosialin, and

siastatin A and B.

References to most of these inhibitors may be found in Walker J M et al,Applied Biochemistry and Biotechnology 38: 141 (1993).

Still other examples of useful inhibitors are:

BASI (see e.g. Rodenburg-K W et al European Journal of Biochemistry 267p. 1019 (2000)), and

T-76 alpha-amylase inhibitor (see Sumitani-j Bioscience biotechnologyand biochemistry 57: 1243 (1993)).

Glycosyl Hydrolases

The glycosyl hydrolases according to the invention are those enzymesacting on glycosidic bonds, which belong to EC 3.2.-.- (as defined inthe Recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology on the Nomenclature andClassification of Enzyme-Catalysed Reactions). It should be noted thatsome of these enzymes are also able to transfer glycosyl residues tooligosaccharides, polysaccharides and other alcoholic acceptors.

Of particular interest for the present invention are enzymes hydrolysingo-glycosyl bonds. These enzymes belong to EC 3.2.1.-. Non-limitingexamples of these are:

-   EC 3.2.1.3 glucan 1,4-alpha-glucosidases, also known as    amyloglucosidases or glucoamylases,-   EC 3.2.1.20 alpha-glucosidases, and-   EC 3.2.1.1 alpha-amylase

An alternative way of classifying enzymes is related to their structure.The CAZy database (see e.g. Davies G., Henrissat B. Structures andmechanisms of glycosyl hydrolases. Structure 3:853-859 (1995); andCoutinho, P. M. & Henrissat, B. (1999) Carbohydrate-active enzymes: anintegrated database approach. In “Recent Advances in CarbohydrateBioengineering”, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., The Royal Society of Chemistry, Cambridge, pp. 3-12) describes thefamilies of structurally-related catalytic and carbohydrate-bindingmodules (or functional domains) of enzymes that degrade, modify, orcreate glycosidic bonds.

Currently, at least 91 families of Glycosyl hydrolases are described. Ofpreferred interest for the present invention are the following families:

Family 13, including the following activities: alpha-amylase (EC3.2.1.1); pullulanase (EC 3.2.1.41); cyclomaltodextringlucanotransferase (EC 2.4.1.19); cyclomaltodextrinase (EC 3.2.1.54);trehalose-6-phosphate hydrolase (EC 3.2.1.93); oligo-alpha-glucosidase(EC 3.2.1.10); maltogenic amylase (EC 3.2.1.133); neopullulanase (EC3.2.1.135); alpha-glucosidase (EC 3.2.1.20); maltotetraose-formingalpha-amylase (EC 3.2.1.60); isoamylase (EC 3.2.1.68); glucodextranase(EC 3.2.1.70); maltohexaose-forming alpha-amylase (EC 3.2.1.98);branching enzyme (EC 2.4.1.18); trehalose synthase (EC 5.4.99.16);4-alpha-glucanotransferase (EC 2.4.1.25); maltopentaose-formingalpha-amylase (EC 3.2.1.-); amylosucrase (EC 2.4.1.4); sucrosephosphorylase (EC 2.4.1.7); malto-oligosyltrehalose trehalohydrolase (EC2.4.1.141); isomaltulose synthase (EC 5.4.99.11).

Family 14, including beta-amylase (EC 3.2.1.2).

Family 15, including the following activities: glucoamylase (EC3.2.1.3); glucodextranase (EC 3.2.1.70).

Family 31, including the following activities: alpha-glucosidase (EC3.2.1.20); glucoamylase (EC 3.2.1.3); sucrase-isomaltase (EC 3.2.1.48)(EC 3.2.1.10); alpha-xylosidase (EC 3.2.1.-); alpha-glucan lyase (EC4.2.2.13); isomaltosyltransferase (EC 2.4.1.-).

Family 57, including the following activites: alpha-amylase (EC3.2.1.1); 4-alpha-glucanotransferase (EC 2.4.1.-); alpha-galactosidase(EC 3.2.1.22).

Family 63, including processing alpha-glucosidase (EC 3.2.1.106).

Of more preferred interest are glycosyl hydrolases belonging to families13 and 15, and most preferably glycosyl hydrolases belonging to family15.

The glycosyl hydrolases of families 1, 2, 3, 5, 7, 10, 11, 12, 13, 16,17, 18, 20, 22, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 42, 51,52, 53, 54, 56, 57, 59, 66, 68, 70, 72, 77, 79, 83, 85 and 86 areretaining glycosyl hydrolases.

Screening

Screening of enzymes has been described in e.g. WO 01/32844. Thescreening method of the invention may be semi or fully automated; it maybe referred to as high throughput screening; it may be capable ofscreening at least 100, preferably at least 500, more preferably atleast 1000, most preferably 5000, and in particular at least 10000glycosyl hydrolases in a continuous operation with no significant humanintervention, except for feeding the setup with miscellaneousconsumables and removing waste; and it may be capable of screening atleast 50, preferably at least 100, more preferably at least 250, mostpreferably 500, and in particular at least 1000 glycosyl hydrolases in24 hours.

In the method of the invention, the glycosyl hydrolases are screened fora property which is dependent on the concentration of the enzyme, inother words a specific property, i.e. a property which has beennormalized by taking the amount of enzyme protein into account. Examplesof specific properties include, but are not limited to, specificactivity (such as activity per mg enzyme or activity per mole) andspecific performance (such as wash performance).

Relating the Screening Result to the Concentration

When carrying out the screening method of the invention, theconcentration of glycosyl hydrolase is determined in step c). Theconcentration must then be related to the screening result by either:

adjusting the concentration of glycosyl hydrolase in each position toessentially the same level and then performing the assay of step d); or

performing the assay of step d) and then correcting the data obtainedwith regard to the concentration of the glycosyl hydrolase, based onknowledge of dosage-response kinetics.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1

Active Site Titration of Glucoamylase Variants and Determination oftheir Specific Activity

Glucoamylase variants may be prepared as described in Sauer J et al.,Biochimica et Biophysica Acta, Vol. 1543 (2), pp. 275-293 (2000)“Glucoamylase: Structure/function relationships, and proteinengineering”;

or as described in Frandsen T P et al., “Increasing the thermalstability and catalytic activity of Aspergillus niger glucoamylase bycombining site specific mutations and directed evolution”, InCarbohydrate Bioengineering, RS—C eds. T T Teeri, B Svensson, H JGilbert and T Feizi, Proceedings of the 4th carbohydrate meeting, 2001.

The Talaromyces emersonii glucoamylase is disclosed in WO 99/28448.

A deep well microtiter plate with 32 Talaromyces emersonii glucoamylasevariants, each grown in three wells, was centrifuged for 5 min at 3000rpm. Obtained culture supernatants were transferred to another deep wellmicrotiter plate. From each well eight aliquots of 40 μl culturesupernatant were transferred to a microtiter plate and mixed with 20 μlof acarbose diluted to varying concentrations (0, 0.0625, 0.125, 0.25,0.5, 1.0, 2.0 and 4.0 μM in 0.2 M sodium acetate buffer, 0.01% TritonX-100, pH 4.5). After incubation for 1 hour at room temperature withagitation, 50 μl substrate solution (10 mM p-nitrophenyl-alpha-D-glucose(pNP-Glu) in 0.2 M sodium acetate buffer, 0.01% Triton X-100, pH 4.5)was added. After 2 hours incubation at room temperature with agitation,the reaction was stopped by adding 50 μl sodium carbonate pH 9.5.Absorbance was read at 405 nm using a microtiter plate reader(SpectraMax Plus, Molecular Devices), and concentrations of culturesupernatants were determined by linear regression with acarboseconcentrations showing residual activity. Highest concentration ofacarbose was in all cases able to inhibit all glucoamylase activity. Nosignificant deviations from a straight line were visible for acarboseconcentrations below the equivalence point indicating that equilibriumbetween inhibitor and enzyme was reached and affinity of inhibitor forenzyme was sufficiently high.

An example of measured and fitted absorbances with the variant Var10 isgiven in Table Acarbose concentration (μM) A₄₀₅ measured A₄₀₅ fitted 02.00 2.00 0.0625 2.06 1.91 0.125 1.80 1.82 0.25 1.57 1.64 0.5 1.17 1.281 0.63 0.56 2 0.04 0.02 4 0.01 0.02Table 2. Measured and fitted absorbances of one of the three deep wellfermentations with the variant Var10. Fitted glucoamylase concentrationwas found from linear regression of absorbances obtained with inhibitorconcentrations from 0 to 1 μM. With 2 and 4 μM inhibitor glucoamylaseactivity was essentially totally inhibited and average of absorbancesobtained with these inhibitor concentrations was assumed to correspondto the background of the assay. As 20 μl inhibitor was mixed with 40 μlculture supernatant this resulted in a fitted glucoamylase concentrationin the culture supernatant of 0.69 μM and a specific activity on pNP-Gluof 52 mOD/min/μM

Specific activity on pNP-Glu was given as negative of slope of activityas function of inhibitor concentration. Results for the determinedconcentrations and specific activities are given in Table 2. Standarddeviation for determined specific activities was on average 7%. TABLE 2Determined concentrations and specific activities of 32 glucoamylasevariants. The results are shown as average ± standard deviation of threewells with same variant. Concentration Specific activity (μM)(mOD/min/μM) WT 0.27 ± 0.05 40 ± 1 Var1 0.33 ± 0.01 38 ± 1 Var2 0.42 ±0.02 30 ± 1 Var3  0.4 ± 0.01 33 ± 2 Var4 0.36 ± 0.02 47 ± 4 Var5 0.35 ±0.03 48 ± 4 Var6 0.47 ± 0.1  51 ± 6 Var7 0.36 ± 0.01 42 ± 4 Var8  0.3 ±0.02 57 ± 3 Var9 0.32 ± 0.01 57.5 ± 0.3 Var10 0.71 ± 0.02 53 ± 1 Var110.38 ± 0.01 49.2 ± 0.3 Var12 0.32 ± 0.04 45 ± 4 Var13 0.37 ± 0.03 41 ± 4Var14 0.36 ± 0.02 41 ± 3 Var15 0.39 ± 0.05 40 ± 3 Var16 0.33 ± 0.01 48 ±3 Var17  0.3 ± 0.01 47 ± 3 Var18 0.31 ± 0.02 43 ± 0 Var19 0.38 ± 0.06 49± 8 Var20 0.38 ± 0.03 47.2 ± 0.8 Var21 0.35 ± 0.01 62 ± 1 Var22 0.33 ±0.01 58 ± 2 Var23 0.31 ± 0.01 62 ± 2 Var24 0.33 ± 0.03 43 ± 2 Var25 0.33± 0.02 58 ± 3 Var26 0.39 ± 0.03 48 ± 2 Var27 0.35 ± 0.04 48 ± 6 Var280.47 ± 0.06 49 ± 4 Var29 0.38 ± 0.04 56 ± 8 Var30 0.41 ± 0.02 58 ± 3Var31 0.39 ± 0.03 62 ± 4

Example 2

Active Site Titration of Xylanase

The synthesized burst active site titrant 2,4-dinitrophenyl2-deoxy-2-flouro-β-D-xylopyranoside was dissolved in Milli Q water. 100μl dissolved titrant was mixed with 50 μl assay buffer (50 mM sodiumacetate, 0.0225% Brij 35, pH 5.0) and 50 μl of a purified sample of thecommercial xylanase Shearzyme (available from Novozymes) diluted inMilli Q water in the wells of a microtiter plate. Final concentrationsof 2,4-dinitrophenyl 2-deoxy-2-flouro-β-D-xylopyranoside were 0.5 mM and1 mM, whereas the xylanase was added to give final absorbances at 280 nmof 13.4, 6.7 and 3.3. After mixing, absorbance was read at 405 nm atroom temperature every 7 min for 30 hours using a SpectraMax Plus(Molecular Devices) microtiter plate reader. After subtraction ofabsorbances read in wells with same concentration of titrant but withoutenzyme, the measurements were fitted to the equation:A ₄₀₅ =B*(1−exp(−(t+LT)*ln(2)/T _(1/2)))+S*(t+LT)

where A₄₀₅ is the absorbance at 405 nm, B is the burst in absorbance at405 nm, t is the time from first measurement of absorbance, LT is thelag time from mixing of the reagents to first measurement of absorbance,T_(1/2) is the half time for the exponential burst phase and S is theslope due to hydrolysis of the enzyme 2-deoxy-2-fluoro-β-D-xylopyranosecomplex.

To calculate the xylanase concentration corresponding to a givenabsorbance burst, a standard curve obtained from absorbances at 405 nmwith known concentrations of 2,4-dinitrophenol in the same volume andbuffer was included.

From the results in Table 3 it is seen that hydrolysis of the enzyme2-deoxy-2-fluoro-β-D-xylopyranose complex (Slope S) is very slowcompared to the initial complex formation liberating 2,4-dinitrophenol(T_(1/2)). Furthermore, the xylanase concentrations calculated from thebursts are close to the ones expected from A280 if the enzyme sample wasentirely pure and fully active being 152 μM, 76 μM and 38 μM with atheoretical molar extinction coefficient of 87870 M⁻¹ cm⁻¹ TABLE 3 Burstactive site titration of the xylanase Shearzyme with the titrant2,4-dinitrophenyl 2-deoxy-2-flouro-β-D-xylopyranoside. Xylanase A₂₈₀13.4 6.7 3.3 13.4 6.7 3.3 Titrant (mM) 1 1 1 0.5 0.5 0.5 Burst B (A₄₀₅)0.90 0.48 0.23 0.84 0.47 0.24 T_(1/2) (h) 4.4 4.2 4.2 8.7 8.6 8.1 SlopeS (A₄₀₅/h) 0.00 0.00 0.00 0.00 0.00 0.00 Xylanase 137 73 35 128 71 37concentration (μM)

Example 3

Synthesis of 2,4-DNP 2-deoxy-2-fluoro-β-D-xylopyranoside

¹H NMR spectra were recorded on a Varian Mercury 400 MHz at 30° C. Flashchromatography was accomplished using a FLASH 40i chromatography modulefrom Biotage. All solvents were purchased from Merck.

3,4-di-O-Acetyl-2-deoxy-2-fluoro-β-D-lyxo- and xylopyranosides 1a,b (ASU14850-046)

3,4-Di-O-acetyl-D-xylal (0.87 g, 4.4 mmol) was dissolved in DMF/H₂O(3:1, 40 mL) and Selectflour (2.5 g, 7.0 mmol) was added. The solutionwas stirred overnight at room temperature before concentrated. Theresidue was dissolved in EtOAc (200 mL) and extracted with water (2×50mL). The aqueous phase was washed with EtOAc (50 mL) and the pooledorganic phases were dried with MgSO4, filtered and concentrated to give0.76 g of crude 1a,b.

2,4-DNP 2-deoxy-2-fluoro-α,β-D-lyxo and xylopyranosides 2a,b,c (ASU14850-049)

The crude product 1a,b (0.40 g, 2.0 mmol) was dissolved in DMF (3 mL)and 2,4-dinitrofluorobenzene FDNB (0.40 g, 2.1 mmol) was added(syringe!) followed by addition of DABCO(1,4-diazabicyclo[2.2.2]-octane, 0.68 g, 6.0 mmol). The solution wasstirred overnight and concentrated. The residue was taken into CHCl₃(100 mL) and extracted with water (2×50 mL) before dried (MgSO₄) andconcentrated to give 0.70 g of crude oil. Chromatography (EtOAc/heptane1:2) gave first pure β-D-lyxo derivative 2b (0.14 g) and second 0.30 gof a mixture of 2a,c. The α-D-lyxo derivative 2c (51 mg) wascrystallized from the mixture by addition of cold EtOAc/heptane (1:2).

2b: ¹H NMR (CDCl₃): 8.75 ppm (d, 1H, DNP), 8.45 ppm (dd, 1H, DNP), 7.50ppm (d, 1H, DNP), 5.90 ppm (d, 1H, J=5 Hz, H-1), 5.54 ppm (m, 1H, H-3),5.05 ppm (dt, 1H, J=5 and 42 Hz, H-2), 5.03 ppm (m, 1H, H-4), 4.23 ppm(dd, 1H, J=2 and 13 Hz, H-5a) and 3.81 ppm (dd, 1H, J=13 and 1 Hz,H-5b), 2.3 ppm (s, 3H, OAc), 2.2 ppm (s, 3H, OAc).

2c: ¹H NMR (CDCl₃, selected data): 8.83 ppm (d, 1H, DNP), 8.48 ppm (dd,1H, DNP), 7.55 ppm (d, 1H, DNP), 5.90 ppm (dd, 1H, J=6 and 3 Hz, H-1),5.32-5.50 ppm (m, 2H, H-3 and H-4), 5.11 ppm (dt, 1H, J=48 and 2 Hz,H-2), 4.10 ppm (dd, 1H, J=11 and 5.5 Hz, H-5a), 3.74 ppm (t, 1H, J=11Hz, H-5b), 2.2 ppm (s, 3H, OAc, 2.1 ppm (2.1 ppm (s, 3H, OAc).

2,4-DNP 2-Deoxy-2-fluoro-β-D-xylopyranoside 3a (ASU 14850-058B)

The mother liquor from the crystallization of 2c containing mainly 2awas deacetylated in 5% HCl—MeOH (prepared by addition of 0.5 mL acetylchloride to 10 mL MeOH) at room temperature overnight. The solution wasconcentrated and evaporated from diethylether (25 mL). The β-D-xyloderivative 3a (27 mg) was selectively crystallized fromMeOH/diethylether/petroleum ether. Mp. 164-165° C. ¹H NMR (CD₃OD,selected data): J_(2,F)=52 Hz.

2,4-DNP 2-Deoxy-2-fluoro-β-D-lyxopyranoside 3b and 2,4-DNP2-Deoxy-2-fluoro-α-D-lyxopyranoside 3c (ASU

The two pure lyxo-derivatives were deacetylated as described above togive the unprotected 3b and 3c.

3b: ¹H NMR (CD₃OD, selected data): 5.84 ppm (dd, 1H, H-1), 4.95 ppm (dt,1H, J_(2,F)=54 Hz, H-2).

3c: ¹H NMR (CD₃OD, selected data): 6.20 ppm (dd, 1H, H-1), 4.88 ppm (dt,1H, J_(2,F)=48 Hz, H-2).

1-9. (canceled)
 10. A method for determining the concentration of aglycosyl hydrolase by active site titration using an inhibitor having aKd which is at least 25 times lower than the concentration of glycosylhydrolase or, when the glycosyl hydrolase is a retaining glycosylhydrolase, using a substrate wherein the rate constant for theglycosylation step is at least 10 times larger than for thedeglycosylation step.
 11. The method of claim 10, wherein Kd is at least100 times lower than the concentration of glycosyl hydrolase.
 12. Themethod of claim 10, wherein the rate constant for the glycosylation stepis at least 100 times larger than for the deglycosylation step.
 13. Themethod of claim 10, wherein the glycosyl hydrolase belongs to family 13glycosyl hydrolase.
 14. The method of claim 10, wherein the glycosylhydrolase belongs to family 14 glycosyl hydrolase.
 15. The method ofclaim 10, wherein the glycosyl hydrolase belongs to family 15 glycosylhydrolase.
 16. The method of claim 10, wherein the glycosyl hydrolasebelongs to family 31 glycosyl hydrolase.
 17. The method of claim 10,wherein the glycosyl hydrolase belongs to family 57 glycosyl hydrolase.18. The method of claim 10, wherein the glycosyl hydrolase belongs tofamily 63 glycosyl hydrolase.
 19. A method of screening for a propertyof a glycosyl hydrolase wherein the property is dependent on theconcentration of the glycosyl hydrolase, comprising the steps of: a)arranging a population of cells expressing glycosyl hydrolases in aspatial array wherein each position of the spatial array is occupied byone or more cells expressing a specific glycosyl hydrolase, b)cultivating the cells in a suitable growth medium, c) determining theconcentration of the glycosyl hydrolase of each position of the spatialarray by active-site titration using an inhibitor having a Kd which isat least 25 times lower than the concentration of glycosyl hydrolase or,when the glycosyl hydrolase is a retaining glycosyl hydrolase, using asubstrate wherein the rate constant for the glycosylation step is atleast 10 times larger than for the deglycosylation step, d) assaying theglycosyl hydrolase of each position of the spatial array for theproperty and relating the result to the concentration.
 20. The method ofclaim 19, wherein the glycosyl hydrolases are expressed recombinantly bythe cells.